1. Field of the Invention
This invention relates generally to the field of gel electrophoresis, and more particularly to an apparatus and method for loading samples into a gel of an electrophoretic gel system.
2. Description of Related Art
Electrophoresis gels are widely used in biotechnology for analyzing biomolecular sample materials such as proteins and nucleic acids. In molecular biology research laboratories, it is well known to use gel electrophoresis to separate and identify sample material based on size, charge and other aspects of the sample material. Biomolecules such as DNA, RNA and protein are commonly separated using this procedure. Electrophoresis involves the migration of electrically charged particles in a gel solution or suspension in the presence of an applied electric field. Samples are inserted or loaded into the gel of an electrophoretic gel system (EGS) and thereafter an electric field is applied to the gel. Each particle in the sample moves toward the electrode having an electrical polarity which is opposite the electrical polarity of the particle. The electrophoretic mobility of a sample particle is inversely proportional to the size of the particle. Various species of a sample may be separated and identified due to differences in electrophoretic mobilities in the gel. The gel also reduces the mixing, or "cross-talk", of various sample particles during the electrophoretic process. Gel electrophoresis thereby facilitates the stable separation and identification of sample material.
Typically, the initial step in manufacturing an EGS is "casting", or polymerization, of the gel. Common techniques used to cast the gel employ a well-forming instrument such as a comb having teeth which extend downwardly toward a sample loading end of the EGS gel. In these systems, the comb teeth are inserted into the EGS gel before polymerization. After the gel polymerizes, the comb is removed from the gel, thereby forming sample wells in the gel where the teeth were inserted. One example of such a sample well-forming instrument is described in U.S. Pat. No. 5,284,565 to Chu, et al. The Chu comb forms sample wells in the EGS gel which taper downwardly toward the gel enclosure. Disadvantageously, it has proven difficult to form structurally consistent sample wells using these well-forming techniques.
FIGS. 1-2 show a prior art electrophoretic gel system. As shown in the electrophoresis apparatus 100 of FIG. 1a, a gel 102 is disposed between two substantially rigid plates 104, 106 which are used to secure the gel 102 in place and to facilitate casting of the gel 102. The gel 102 typically comprises agarose or polyacrylamide. A well-forming comb 108 having a plurality of teeth or fingers 110 is used to form sample wells in the gel 102. As shown in FIG. 1a, the comb 108 is inserted between the plates 104, 106 into an upper edge of the gel 102 before the gel polymerizes. As shown in FIG. 1b, after the gel polymerizes, the comb 108 is removed from the upper edge of the gel 102. A plurality of sample wells 112 are thus formed between the plates 104, 106 in the upper edge of the gel 102. Liquid samples are then introduced into the sample wells 112 wherein they form individual sample pools, each separated by partitions 114 formed in the upper edge of the gel 102. To facilitate the electrophoresis process, the apparatus 100 is immersed in an electrode buffer solution. The buffer solution may be liquid, gel or paste. As shown in FIG. 1b, the apparatus 100 is immersed in the electrode buffer solution so that the level of the solution 116 is slightly below the top edge of the plate 104. After the liquid samples are introduced into the sample wells 112, an electric field is applied to the electrode buffer solution so that electric current passes through the buffer solution, sample material, and gel 102. The samples in the sample wells 112 then migrate toward the bottom of the gel and separate due to their differing migration characteristics.
FIGS. 2a and 2b show a prior art horizontal EGS. The horizontal EGS 200 includes a gel casting structure which has two substantially rigid removable dams 202 shaped to be received by the EGS 200. Before solidification or congelation of a gel 204, the gel 204 is poured into the EGS 200 and is cast by the EGS walls and the removal dams 202. The comb fingers 110 (FIGS. 1a and 1b) are then inserted into the gel solution 204 before the gel congeals. After the gel congeals, the fingers 110 of the comb 108 are removed from the gel 204, as shown in FIG. 2b. The comb 108, and specifically the teeth 110, form sample wells 112 in the gel 204. Sample material is then typically manually inserted into the wells 112 using a pipette. One of the drawbacks of this sample insertion technique is that it requires a high degree of technical skill and dexterity. Consequently, the insertion of sample material into the wells is a time-intensive process which is prone to error and inadvertent spillage of sample material into the buffer solution. Therefore, there is a need for an improved gel electrophoresis method and apparatus which facilitates the loading of samples into an EGS.
As shown in FIG. 2b, the gel is immersed in an electrode buffer solution for electrophoretic separation. Similar to the apparatus of FIG. 1, the electrode buffer solution is filled to a level 116 which is sufficient to immerse the gel 204. The EGS 200 typically includes connections to a power supply (not shown). As shown in FIG. 2b, a cathode wire 206 is immersed in the electrode buffer solution at an upper end of the EGS 200 proximate the sample well end of the gel 204. An anode wire 208 is immersed in the buffer solution at a bottom end of the EGS 200 near a distal end of the gel 204. Thereafter, as described above, an electric field is typically applied to the buffer solution and the samples previously inserted into the sample wells 112 separate due to the electrophoretic effect.
Another prior art gel electrophoresis system and sample loader is disclosed in U.S. Pat. No. 5,275,710 issued to Gombocz et al. Gombocz teaches a sample loader or applicator which has a plurality of applicator channels covered by an absorbent material (such as a wick) which prevents the uninhibited flow of sample material into the gel. When the wicks are loaded with a measured amount of sample material, the applicator is lowered into the gel so that the wicks contact the gel, which allows the sample material to perfuse through the wicking material and into the gel. Disadvantageously, the applicator taught by Gombocz allows sample material to diffuse into the gel and surrounding buffer before the gel is activated by applying an electric field. Therefore, a need exists for a sample loading apparatus and method for loading samples into an EGS which allows samples to be easily, quickly and accurately loaded into the gel yet which inhibits the electrophoretic separation of the samples until activation by the release mechanism.
One of the problems associated with the prior art sample loading apparatuses is the potential for inadvertent cross-contamination via the buffer solution. As shown in FIG. 1b, if the samples overflow the sample wells 112, the buffer solution becomes contaminated with sample material, which may lead to erroneous results due to mixing of sample material between sample wells. Therefore, there is a need for a method and apparatus for loading sample material into an EGS which inhibits the diffusion of sample material into the gel or buffer before electrophoretic action commences.
Another drawback associated with the prior art EGS sample loading devices is due to the incomplete polymerization of certain types of gel material such as polyacrylamide due to the presence of a polymerization inhibitor. For example, due to the presence of oxygen, the area surrounding the comb teeth in prior art EGS loaders is often incompletely polymerized. Also, because the sample wells 112 are typically spaced in very close proximity to each other, the walls or partitions 114 are often quite fragile and as a result are easily damaged or destroyed. Another drawback associated with the prior art methods and apparatuses for loading samples into an EGS is the difficulty in removing undesirable materials such as urea from the sample wells after use.
Therefore, there is a need for an improved apparatus and method for loading sample material into a gel of an EGS. The present invention provides such an apparatus and method.